Complete control of HIV-1 remains an elusive goal despite the significant impact of antiretroviral drugs. Emergence of drug-resistant HIV-1 variants and the reactivation of latent HIV-1 reservoirs remain formidable obstacles. Furthermore, the 2',5'-oligoadenylate synthetase (2-5OAS)/RNase L and p68 kinase (PKR) innate antiviral defense pathways are inhibited following HIV-1 infection. The working hypothesis of this project is that restoration of these antiviral defense pathways can provide an effective therapeutic approach to inhibit HIV-1 replication. We will apply two strategies to achieve this goal: 1) intracellular immunization of PKR and 2-5OAS transgenes to protect cells against HIV-1 infection and 2) nuclease-resistant, non-toxic 2-5A agonists that activate RNase L and induce interferon and chemokine expression. The specific aims are: 1. To determine the anti-HIV-1 effects of expression of the antiviral transgenes, PKR and 2-5OAS, in transduced CD34+ hematopoietic stem cells and their differentiated T cell and monocyte progeny following delivery by HIV-1 based self-inactivating lentiviral vectors (SIN LVs). SIN LVs can transduce non-dividing and dividing cells with increased biosafety and enhanced transgene expression. As components of the innate antiviral defense pathways, the PKR and 2-5OAS gene products are not subject to host immune surveillance or affected by mutations in HIV-1. 2. To determine the in vitro anti-HIV effects of two select 2-5A agonists in resting CD4+ T lymphocytes from viremic and aviremic HIV-1 seropositive patients. These 2-5A agonists circumvent HIV-1 induced blockades in antiviral defense by mechanisms of action distinct from other anti HIV-1 strategies. 3. To inhibit replication of reactivated HIV-1from persistently infected resting CD4+ T cells by transduction with SIN LVs encoding PKR and 2-5OAS transgenes. Combination experiments of SIN LV- transduced cells with 2-5A agonists or approved anti-HIV-1 drugs will be conducted with the goal of additive or synergistic anti-HIV-1 activity. These anti-HIV-1 strategies, which utilize the innate antiviral defense pathways, offer a new therapeutic approach to the inhibition of HIV-1 replication.